Regulation of Hip1r by epsin controls the temporal and spatial coupling of actin filaments to clathrin-coated pits

Abstract

Recently, it has become clear that the actin cytoskeleton is involved in clathrin-mediated endocytosis. During clathrin-mediated endocytosis, clathrin triskelions and adaptor proteins assemble into lattices, forming clathrin-coated pits. These coated pits invaginate and detach from the membrane, a process that requires dynamic actin polymerization. We found an unexpected role for the clathrin adaptor epsin in regulating actin dynamics during this late stage of coated vesicle formation. In Dictyostelium cells, epsin is required for both the membrane recruitment and phosphorylation of the actin- and clathrin-binding protein Hip1r. Epsin-null and Hip1r-null cells exhibit deficiencies in the timing and organization of actin filaments at clathrin-coated pits. Consequently, clathrin structures persist on the membranes of epsin and Hip1r mutants and the internalization of clathrin structures is delayed. We conclude that epsin works with Hip1r to regulate actin dynamics by controlling the spatial and temporal coupling of actin filaments to clathrin-coated pits. Specific residues in the ENTH domain of epsin that are required for the membrane recruitment and phosphorylation of Hip1r are also required for normal actin and clathrin dynamics at the plasma membrane. We propose that epsin promotes the membrane recruitment and phosphorylation of Hip1r, which in turn regulates actin polymerization at clathrin-coated pits.

Fig. 1.

Actin polymerization functions in the late stages of clathrin-mediated endocytosis in Dictyostelium. (A) TIRF images of the membrane of wild-type cells expressing clathrinRFP (clathrin) and limEΔcoilGFP (actin). Scale bar: 5 μm. (B) Time-lapse images of the individual clathrinRFP punctum (clathrin) identified in A from appearance to internalization accompanied by a punctum of actin labeled by limEΔcoil (actin). (C) Treatment with the actin depolymerizing drug cytochalasin A leads to an accumulation of clathrin puncta on the plasma membrane. Epifluorescence images from surface and middle focal planes of wild-type cells expressing clathrinRFP. Cells treated with cytochalasin A (+cytA) show increased numbers of clathrin puncta on the plasma membrane, 0.89±0.04 puncta per μm, compared to control cells (−cytA), 0.39±0.02 puncta per μm. Mean ± s.e.m., n=30 cells. Scale bar: 5 μm. (D) Treatment with the actin depolymerizing drug Latrunculin A treatment causes clathrin pits to persist on the membrane. TIRF time-lapse images of cells expressing clathrinGFP (clathrinGFP) with (+LatA) and without (−LatA) treatment. (E) Quantification of clathrin pits colocalizing with epsin and AP2 simultaneously in the presence (60±2%; n=12 cells) and absence (61±4%; n=12 cells) of cytochalasin A treatment. (F) Quick-freeze deep etch scanning electron micrograph of assembled clathrin lattices in wild-type cells treated with Latrunculin A (WT + LatA) show that the morphology of clathrin lattices is unaffected. (G) Latrunculin A treatment causes an accumulation of invaginated clathrin-coated pits. Wild-type cells expressing clathrinGFP were treated with Latrunculin A and imaged under IRM and epifluorescence microscopy (GFP). Arrows indicate colocalization between clathrin signal and deeply invaginated pits. All quantification was performed on cells from three independent experiments for each condition.

Fig. 2.

Hip1r and epsin, but not AP180, are required for normal clathrin and actin dynamics at the plasma membrane. (A) Clathrin puncta persist at the membrane of epsin and Hip1r-null cells. Time-lapse TIRF images of wild-type (WT), AP180-null (AP180−), epsin-null (epsin−), and Hip1r-null (Hip1r−) cells expressing clathrinRFP. (B) Quantification of the lifetime of clathrin puncta identified at the beginning of TIRF time-lapse acquisition. Wild-type (39±2 seconds, n=49; AP180-null (AP180−) 30±2 seconds, n=35; epsin-null (epsin−) 70±6 seconds, n=31; and Hip1r-null (Hip1r−) 68±4 seconds, n=29. (C) Representative plot showing the fluorescence intensity of a wild-type clathrinRFP (clathrin) punctum over time; 1, 2 and 3 mark the assembly, plateau and internalization phases, respectively. (D) Quantification of the average lifetime (seconds) of clathrin puncta from wild-type (n=20), AP180-null (n=20), epsin-null (n=16), and Hip1r-null (n=20) cells. Cells are in the assembly phase (1) with normalized intensities of 25–75 a.u.; plateau phase (2) with intensities above 75 a.u.; and internalization phase (3) with intensities of 75–25 a.u. (E) Average plots of the intensity of clathrinRFP (clathrin) puncta over time, with the accompanying actin puncta as labeled by limEΔcoilGFP (actin) in wild-type, AP180-null, epsin-null, and Hip1r-null cells; n=16–20 per cell line. (F) Time-lapse TIRF images of individual clathrin and actin puncta in wild-type, AP180-null, epsin-null, and Hip1r-null cells coexpressing clathrinRFP (clathrin) and limEΔcoilGFP (actin). (G) Quantification of laterally mobile actin puncta as labeled by limEΔcoilGFP: wild-type 11±8%, n=35 puncta on 5 cells; AP180-null 22±3%, n=39 puncta on 6 cells; epsin-null 56±6%, n=49 puncta on 5 cells; and Hip1r-null 53±6%, n=51 puncta on 6 cells. All values are mean ± s.e.m. All quantification was performed on cells from three independent experiments for each condition.

Fig. 3.

Disrupting actin polymerization affects Hip1r localization but not phosphorylation in epsin-null cells. (A) Epifluorescence images from a middle focal plane of wild-type cells (−CytA) and cells treated with cytochalasin A (+CytA), immunostained with anti-Hip1r (α-Hip1r) antibodies. Scale bar: 5 μm. (B) Epifluorescence images from a middle focal plane of epsin-null cells and epsin-null cells treated with cytochalasin A, immunostained with anti-Hip1r antibodies. Note the increased Hip1r localization to the membrane in epsin-null cells treated with cytochalasin A. Scale bar: 5 μm. (C) Quantification of Hip1r localization in wild-type (WT) and epsin-null cells (epsin−) with and without cytochalasin A treatment: WT −cytA, 0.48 ±0.03 puncta per μm; WT +cytA, 0.61±0.02 puncta per μm; epsin− −cytA, 0.16±0.01 puncta per μm; epsin− +cytA, 0.61±0.02 puncta per μm. All values are mean ± s.e.m., n=30 cells from three independent experiments for each condition. (D) Epifluorescence images from a surface focal plane of wild-type and epsin-null cells expressing clathrinGFP (clathrin), treated with cytochalasin A, and immunostained for Hip1r. Scale bar: 5 μm. (E) Quantification of colocalization between Hip1r and clathrin in wild-type and epsin-null cells treated with cytochalasin A: wild-type 76±5%, n=14 cells; Hip1r-null 67±2%, n=20 cells. (F) Western blots of whole cell lysates from wild-type, epsin-null, and epsin-null cells expressing epsinGFP (epsin− + epsinGFP) with or without cytochalasin A treatment. Blots were probed with anti-Hip1r antibodies. Note that cytochalasin A treatment does not induce a phosphorylated species of Hip1r in epsin-null cells.

Fig. 4.

EpsinT107A does not rescue the Hip1r and actin-related phenotypes of epsin-null cells. (A) Epifluorescence images from a middle focal plane of epsin-null cells expressing epsin^WTGFP (WT), epsin^R65A/K78AGFP (R65A/K78A), or epsin^T107AGFP (T107A) and immunostained for Hip1r (α-Hip1r). Scale bar: 5 μm. (B) Quantification of Hip1r puncta at the membrane of epsin-null cells expressing epsin^WTGFP (epsinWT, 0.40±0.03 puncta per μm), epsin^R65A/K78AGFP (epsinR65A/K78A, 0.15±0.01 puncta per μm) epsin^T107AGFP (epsinT107A, 13±0.01 puncta per μm); n=30 cells from three independent experiments for each cell line. (C) Immunoblots of whole cell lysates from wild-type (WT) and epsin-null (epsin−) cells, and epsin-null cells expressing epsin^WTGFP (epsin− + epsin^WT), epsin^R65A/K78AGFP (epsin− + epsin^R65A/K78A), or epsin^T107AGFP (epsin− + epsin^T107A). Blots were probed with anti-Hip1r (α-Hip1r) or anti-GFP (α-GFP) antibodies. Center row indicates whole-cell lysates treated with calf intestinal phosphatase (CIP). (D) Time-lapse TIRF images of epsin-null cells coexpressing limEΔcoilGFP and either epsin^WTGFP or epsin^T107AGFP. (E) Quantification of lateral mobility of actin puncta labeled by limEΔcoilGFP in epsin-null cells expressing either epsin^WTGFP (21±2%, n=23 puncta on three cells) or epsin^T107AGFP (43±7%, n=30 puncta on three cells).